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Biol Open
2018 Jul 23;77:. doi: 10.1242/bio.034926.
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Location and functions of Inebriated in the Drosophila eye.
Borycz J
,
Ziegler A
,
Borycz JA
,
Uhlenbrock G
,
Tapken D
,
Caceres L
,
Hollmann M
,
Hovemann BT
,
Meinertzhagen IA
.
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Histamine (HA) is a neurotransmitter in arthropod photoreceptors. It is recycled via conjugation to β-alanine to form β-alanylhistamine (carcinine). Conjugation occurs in epithelial glia that surround photoreceptor terminals in the first optic neuropil, and carcinine (CA) is then transported back to photoreceptors and cleaved to liberate HA and β-alanine. The gene Inebriated (Ine) encodes an Na+/Cl--dependent SLC6 family transporter translated as two protein isoforms, long (P1) and short (P2). Photoreceptors specifically express Ine-P2 whereas Ine-P1 is expressed in non-neuronal cells. Both ine1 and ine3 have significantly reduced head HA contents compared with wild type, and a smaller increase in head HA after drinking 1% CA. Similarly, uptake of 0.1% CA was reduced in ine1 and ine3 mutant synaptosomes, but increased by 90% and 84% respectively for fractions incubated in 0.05% β-Ala, compared with wild type. Screening potential substrates in Ine expressing Xenopus oocytes revealed very little response to carcinine and β-Ala but increased conductance with glycine. Both ine1 and ine3 mutant responses in light-dark phototaxis did not differ from wild-type. Collectively our results suggest that Inebriated functions in an adjunct role as a transporter to the previously reported carcinine transporter CarT.
Fig. 1. Expression of P1 and P2 isoforms in the compound
eye. Oregon R wild-type flies labelled with antibodies against carcinine (green channel), and the P1 (red channel) and P2 (blue channel) protein isoforms of Inebriated. (A) In the compound eye P1 and P2 signals mostly fail to overlap. (B) Enlarged view of distalretina. (C) In the retina P2 (blue) strongly labels pseudocone cavities. (D) Inset: transversely sectioned ommatidia. P1 labels secondary and tertiary pigment cells (red, arrow) whereas P2 labels mostly photoreceptors (blue, arrowhead). Scale bars: 50 µm (A); 20 µm (B).
Fig. 2. Expression of P1 and P2 isoforms in the optic lobe. Ine-P1 (A,B,C; magenta) and Ine-P2 (D,E,F; blue) co-expression with Gal4 driven GFP expression (green), reveals specific types of optic lobe glia (Edwards et al., 2012). (A) Ine-P1 co-expresses with GFP in the distal satellite glia (dsg) but not in the proximal satellite glia (psg). (B) Ine-P1 co-expresses with GFP in the distal satellite glia (dsg) but not in the marginal glia (mg). (C) Ine-P1 co-expresses with GFP in the pseudocartridge glia (psug). (D) Ine-P2 co-expresses with GFP in proximal satellite (psg) and marginal (mg) glia. (E) Ine-P2 co-expresses with GFP in marginal glia (mg). (F) Ine-P2 co-expresses with GFP in pseudocartridge (psug) and proximal satellite (psg) glia. (G) Ine-P1 co-expression with GFP in the distal satellite glia (dsg), enlarged from A. (H) Transverse section of lamina cartridges. Ine-P1 co-expresses with epithelial glia marker (arrow) and Ine-P2 expresses in the photoreceptors (arrowhead). (I) Enlarged view of cartridge array. Glia are labelled with the following abbreviations (from distal to proximal): fg, fenestrated glia; psug, pseudocartridge glia; dsg, distal satellite glia; psg, proximal satellite glia; eg, epithelial glia; mg: marginal glia. Scale bars: 20 µm (F, for A–F), 20 µm (H) and 5 µm (I).
Fig. 3. Expression of the long (Ine-P1; magenta) and short (Ine-P2; green) isoforms of Inebriated in alternate layers of glia of the fly's optic lamina. Ine-P1 is expressed in the fenestrated glia (fg), the distal (d) region of the satellite glia (sg), and the epithelial glia (eg). Ine-P2 is expressed in the pseudocartridge glia (pg), proximal (p) satellite glia (sg), and marginal glia (mg). Successive layers from distal to proximal are: the basement membrane (bm), fenestrated layer (fl), nuclear layer (nl), plexiform layer (pl) and photoreceptors axons (ra). Figure based on Saint Marie and Carlson (1983); the distinction between two layers of satellite glia (sg), proximal (p) and distal (d), was made according to Edwards and Meinertzhagen (2010).
Fig. 4. Immunoreactivity to HA, CA and β-Ala are all reduced in specific cell types in ine1 mutant flies compared with Oregon R wild type (wt), indicating the selective reductions in histamine and its two major metabolites. The reduction was greatest in the lamina, and the difference most distinct with anti-carcinine labelling in the marginal glia (arrows). Scale bar: 50 µm (β-alanine in ine1).
Fig. 5. Head HA (A), β-Ala (B) and CA (C) are all reduced in ine1 and ine3 mutants, compared with Oregon R wild-type flies (wt), confirming reductions in immunolabelling seen in
Fig. 4. Values differ statistically at P<0.05 (+) or P<0.01 (++), ANOVA followed by Tukey's HSD test (mean±s.d.; n=12/group).
Fig. 6. (A) Head HA content from flies after drinking solutions of carcinine (1%) suspended in 4% glucose for 12 h. The heads of both ine1 and ine3 mutant flies accumulate significantly less histamine after drinking the carcinine solution than Oregon R control wild-type flies (mean±s.d.; n=7/group). (B) Head accumulation of tritiated β-Alanine (β-Ala) after drinking for 40 min 25% [3H] β-alanine (37 MBq l−1 and 1850 GBq mmol−1 suspended in 4% glucose. Both ine mutants accumulate in their heads twice as much tritiated β-Ala as do Oregon R wild-type control flies (wt). Injected volume represents the radioactivity in a single head and is shown as counts per minute (CPM). (C–E) Fly synaptosome fractions incubated for 1 h in either 0.05% HA or 0.05% β-Ala or 0.1% CA suspended in fly saline (each group n=7). (C) The uptake of 0.05% HA into the synaptosomes of wt and ine mutant fly heads showed no differences; whereas (D) the uptake of 0.05% β-Ala was significantly increased in ine flies; and, in contrast (E), the uptake of 0.1% CA is strongly reduced in both ine mutants. (D–E) Values differ statistically at P<0.01 (++), ANOVA followed by Tukey's HSD test (C–E, mean±s.d.; n=7/group).
Fig. 7. Effects of β-Ala and CA on transmembrane conductance of Xenopus laevis oocytes transfected with Inebriated RNA. (A) Oocytes expressing Ine-P1::GFP or Ine-P2::GFP on the left, next to uninjected control oocytes. (B,C) Current recordings from Xenopus oocytes expressing Ine-P1or water-injected controls held in solutions of β-alanine or carcinine at the given concentrations. (B) Representative traces show currents induced by applying various concentrations of β-alanine and carcinine, and glycine. (C) Glycine generates a significant current in oocytes transfected by transcripts for both the long (Ine-P1) and short (Ine-P2) Inebriated isoforms. Oocytes transfected by the transcript of the long (Ine-P1) protein isoform exhibit dose-dependent glycine currents over a range from 7 mM to 100 mM, relative to control oocytes (right).
Fig. 8. Phototactic index for ine mutant Drosophila relative to hdc flies, which lack HA. (A) Image of phototaxis assay equipment used to quantify Drosophila phototaxis in the light-dark paradigm. (B,B′) Diagram of the T-maze setups used to record phototaxis. In the light-dark paradigm (B) the lit arm (clear glass tube shown as an open rectangle) was illuminated by two blue-white LEDs (Luxeon Star/O, LXHK- WE8). The other tube (dark rectangle) was opaque. In the blue-green choice paradigm (B′) both tubes were clear, illuminated in one by four blue LEDs (467 nm; RL5-B55515 SuperbrightLeds) and in the other by four green LEDs (525 nm; RL5-G13008 SuperbrightLeds). Flies are manipulated in three slider positions of the specimen chamber: load (1), intermediate (2) and testing (3). The dimensions shown are not in proportion. (C) Wild-type OR flies show a strong preference for the lit arm, whereas most hdcJK910 flies fail to move into either the lit or dark arm. L–D phototactic indices calculated from ine mutant performances differ significantly from blind hdcJK910 flies, at P<0.01 (++) followed by Tukey's HSD test, but do not differ from Oregon R wild-type control flies in a light-dark paradigm. (D) ine1 and ine3 show a smaller preference for the blue-lit arm compared with wild type but the differences are not statistically significant. (C–D) mean±s.d.; n=8/group.
Agullo,
Beta-alanine transport in synaptic plasma membrane vesicles from rat brain. Efflux, exchange and stoichiometry.
1986, Pubmed
Agullo,
Beta-alanine transport in synaptic plasma membrane vesicles from rat brain. Efflux, exchange and stoichiometry.
1986,
Pubmed
Aust,
Alternative tasks of Drosophila tan in neurotransmitter recycling versus cuticle sclerotization disclosed by kinetic properties.
2010,
Pubmed
Borycz,
The determination of histamine in the Drosophila head.
2000,
Pubmed
Borycz,
The metabolism of histamine in the Drosophila optic lobe involves an ommatidial pathway: β-alanine recycles through the retina.
2012,
Pubmed
Borycz,
tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals.
2002,
Pubmed
Borycz,
Drosophila ABC transporter mutants white, brown and scarlet have altered contents and distribution of biogenic amines in the brain.
2008,
Pubmed
Bosch,
Dscam2 affects visual perception in Drosophila melanogaster.
2015,
Pubmed
Burg,
Genetic and molecular identification of a Drosophila histidine decarboxylase gene required in photoreceptor transmitter synthesis.
1993,
Pubmed
Burg,
Drosophila rosA gene, which when mutant causes aberrant photoreceptor oscillation, encodes a novel neurotransmitter transporter homologue.
1996,
Pubmed
Chaturvedi,
Drosophila Vision Depends on Carcinine Uptake by an Organic Cation Transporter.
2016,
Pubmed
Chiu,
The transporter-like protein inebriated mediates hyperosmotic stimuli through intracellular signaling.
2000,
Pubmed
,
Xenbase
Edwards,
The functional organisation of glia in the adult brain of Drosophila and other insects.
2010,
Pubmed
Edwards,
Organization and metamorphosis of glia in the Drosophila visual system.
2012,
Pubmed
Frenkel,
Organization of Circadian Behavior Relies on Glycinergic Transmission.
2017,
Pubmed
Fykse,
Amino acid neurotransmission: dynamics of vesicular uptake.
1996,
Pubmed
Gavin,
The role of carcinine in signaling at the Drosophila photoreceptor synapse.
2007,
Pubmed
Gorostiza,
A decision underlies phototaxis in an insect.
2016,
Pubmed
Hardie,
Is histamine a neurotransmitter in insect photoreceptors?
1987,
Pubmed
Heisenberg,
Separation of receptor and lamina potentials in the electroretinogram of normal and mutant Drosophila.
1971,
Pubmed
Hotta,
Abnormal electroretinograms in visual mutants of Drosophila.
1969,
Pubmed
Huang,
In vivo properties of the Drosophila inebriated-encoded neurotransmitter transporter.
2002,
Pubmed
Huang,
The Drosophila inebriated-encoded neurotransmitter/osmolyte transporter: dual roles in the control of neuronal excitability and the osmotic stress response.
2002,
Pubmed
Kinjo,
Evolutionary history of the GABA transporter (GAT) group revealed by marine invertebrate GAT-1.
2013,
Pubmed
Luan,
The putative Na⁺/Cl⁻-dependent neurotransmitter/osmolyte transporter inebriated in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis.
2015,
Pubmed
Melzig,
Genetic depletion of histamine from the nervous system of Drosophila eliminates specific visual and mechanosensory behavior.
1996,
Pubmed
Pantazis,
Distinct roles for two histamine receptors (hclA and hclB) at the Drosophila photoreceptor synapse.
2008,
Pubmed
Richardt,
Ebony protein in the Drosophila nervous system: optic neuropile expression in glial cells.
2002,
Pubmed
Rudnick,
The SLC6 transporters: perspectives on structure, functions, regulation, and models for transporter dysfunction.
2014,
Pubmed
Saint Marie,
Glial membrane specializations and the compartmentalization of the lamina ganglionaris of the housefly compound eye.
1983,
Pubmed
Sarthy,
Histamine: a neurotransmitter candidate for Drosophila photoreceptors.
1991,
Pubmed
Soehnge,
A neurotransmitter transporter encoded by the Drosophila inebriated gene.
1996,
Pubmed
Stenesen,
The carcinine transporter CarT is required in Drosophila photoreceptor neurons to sustain histamine recycling.
2015,
Pubmed
Stern,
Identification and characterization of inebriated, a gene affecting neuronal excitability in Drosophila.
1992,
Pubmed
Stuart,
The dynamics of signaling at the histaminergic photoreceptor synapse of arthropods.
2007,
Pubmed
Tapken,
A plant homolog of animal glutamate receptors is an ion channel gated by multiple hydrophobic amino acids.
2013,
Pubmed
,
Xenbase
Vandenberg,
Mechanisms of glutamate transport.
2013,
Pubmed
Xu,
Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors.
2015,
Pubmed
Yager,
Control of Drosophila perineurial glial growth by interacting neurotransmitter-mediated signaling pathways.
2001,
Pubmed
Ziegler,
Activity and coexpression of Drosophila black with ebony in fly optic lobes reveals putative cooperative tasks in vision that evade electroretinographic detection.
2013,
Pubmed
